Clinical phenotype and laboratory characteristics of 93 patients with congenital fibrinogen disorders from unrelated 36 families

Background Congenital fibrinogen disorders (CFDs) are rare bleeding disorders (RBDs) caused by mutations in 1 of the 3 fibrinogen genes (FGA, FGB, and FGG). Objectives To investigate the clinical phenotype, laboratory features, diagnosis, treatment, and prognosis of CFDs. Methods Clinical data of 93 subjects with CFDs identified from June 2018 to December 2023 were retrospectively analyzed. Results Among the 93 patients, there were 46 males (49.5%) and 47 females (50.5%), with a median age of 23 years. Fifty-three of 93 (57%) subjects experienced bleeding, 3/93 (3.2%) experienced thrombosis, and 37/93 (39.8%) were asymptomatic. Females were more prone to experience bleeding (P < .0001). The 93 patients exhibited prolonged thrombin time, significantly decreased fibrinogen activity (Fg:C), and normal or decreased fibrinogen antigen. The 93 patients included 3 with hypofibrinogenemia, 16 with hypodysfibrinogenemia, and 74 with dysfibrinogenemia. Among the 53 patients with bleeding, bleeding episodes were identified in 3.8% (2/53), 20.8% (11/53), and 75.5% (40/53) patients with hypofibrinogenemia, hypodysfibrinogenemia, and dysfibrinogenemia, respectively. Genetic analysis was performed on 22 cases from 8 pedigrees, revealing 10 mutations, including 1 novel splice mutation. Twenty-eight (30.1%) subjects received replacement therapy to treat or prevent bleeding, consisting of 8 fresh frozen plasma transfusions, 3 packing and suture treatment, and 61 fibrinogen infusions. Conclusion Most patients with CFDs have mild or no bleeding symptoms. Fg:C combined with fibrinogen antigen and pedigree investigation can improve the feasibility and accuracy of diagnosis of CFDs. The severity of bleeding symptoms was negatively correlated with Fg:C.

Congenital fibrinogen disorders (CFDs) are rare bleeding disorders (RBDs) caused by mutations in 1 of the 3 fibrinogen genes (FGA, FGB, and FGG) [3,4].However, recent data from the World Hemophilia Federation survey indicate that CFDs account for 8% of all RBDs, and their incidence is increasing compared with other autosomal heritable RBDs [5,6].Previous studies on CFDs mainly consisted of case reports or limited family reports, making it difficult to establish correlations between laboratory features, clinical phenotype, treatment, and prognosis due to their rarity.
Herein, we performed a retrospectively analysis of 93 subjects from 36 unrelated families with CFDs to explore the clinical phenotype, laboratory features, treatment, and prognosis of CFDs.

| M E T H O D S 2.1 | Identification of study subjects and clinical evaluation
We recruited child probands with reduced fibrinogen activity (Fg:C) and prolonged thrombin time (TT) without adequate clinical explanation.Those child probands were patients from the Children's Hospital of Zhejiang University, School of Medicine (Hangzhou, China), between June 2018 and December 2023.We identified potential subjects including child probands and their family members through pedigree investigation.We determined if they fulfill the following inclusion criteria: 1) decreased Fg:C, 2) normal or prolonged TT, and 3) normal liver and kidney functions.The cutoff thresholds used to define "normal," "prolonged," and "reduced" test results were the 95% reference intervals for each test derived at each laboratory.Those with identifiable causes of acquired CFDs [7] and those family members with normal coagulation function were excluded.
All potential subjects underwent clinical evaluation after obtaining written informed consent.We recorded the historical clinical symptoms of bleeding or thrombosis by inpatient/outpatient medical history, patient interview, and telephone questionnaire.The clinical information of bleeding includes the presence or absence of bleeding symptoms, type of bleeding, bleeding frequency, quantity of bleeding, and the level of medical attention and treatment required for each.Clinical bleeding manifestations were quantified using the consensus International Society on Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) [8,9].
All procedures were conducted in accordance with the Declaration of Helsinki, and the study was approved by the Ethics Committee of Zhejiang University School of Medicine with the ethics board number 2023-IRB-0189-P-01.

| Coagulation tests
Venous blood samples were collected in tubes with 0.109 mol/L sodium citrate.All blood samples were centrifuged at 1500 g for 15 minutes to produce platelet-poor plasma.The prothrombin time (PT), activated partial thromboplastin time (aPTT), and TT were detected by the 1-stage clotting method.The Fg:C and fibrinogen antigen (Fg:Ag) were tested by the Clauss [10] and PT-derived methods, respectively.PT-derived method is a clinical practical method and correlates well with immunologic assays, the gold standard measure of Fg:Ag [11][12][13].In the study, the normal reference values for all coagulation tests in children were as follows: PT, 9.0 to 14.0 seconds; APTT, 23.0 to 38.0 seconds; TT, 15.0 to 22.0 seconds; and Fg, 1.8 to 4.0 g/L); the normal reference values in adults were as follows: PT, 9.0 to 14.0 seconds; APTT, 22.0 to 35.0 seconds; TT, 14.0 to 21.0 seconds; and Fg, 2.0 to 4.0 g/ L. All these assays were performed by CS-5100 automated coagulation analyzer (Sysmex Corporation).All operations were conducted following the manufacturer's protocols.

| The classification of CFDs
According to the recommendation [5,[14][15][16] of domestic and foreign experts on the diagnosis and classification of CFDs, CFDs include afibrinogenemia (complete absence of fibrinogen), hypofibrinogenemia (Hypo-; proportional decrease of functional and antigenic fibrinogen levels), dysfibrinogenemia (Dys-; decreased functional and normal antigenic fibrinogen levels), and
• We analyzed the clinical data of 93 patients with CFDs from 36 families.
• Most patients with CFDs have mild or no bleeding symptoms.
• The severity of bleeding was negatively correlated with fibrinogen activity levels.hypodysfibrinogenemia (Hypodys-; discrepant decrease of functional and antigenic fibrinogen levels).

| DNA sequence analysis
DNA specimens for gene analysis were extracted from the anticoagulant blood samples using DNA Extraction Kit (Tiangen) according to the manufacturer's protocol.The primer sequences were designed according to the Fg gene sequence from the GenBank (FGA:NM_021871, FGB:NM_005141, and FGG:NM_021870), covering all exon regions of Fg gene and its flanks sequence.All the polymerase chain reaction (PCR) amplifications were carried out in a thermal circulator (Eppendorf).A total volume of PCR system was 25 μL, including 2×Es Taq MasterMix of

| Statistical analysis
Normally distributed variables are presented as mean ± SD, while nonnormally distributed variables are presented as medians and IQR (25th-75th percentiles).Normally distributed variables were analyzed by independent sample t test, while nonnormally distributed variables were analyzed by the nonparametric Mann-Whitney U-test.Categorical variables were compared with use of chi-squared analysis.The relationships between 2 variables were analyzed using the Spearman correlation analysis.Data were analyzed using SPSS 20.0 (SPSS), and a 2-sided P value of <.05 was considered statistically significant.
For women, Fg:C levels were slightly lower in patients with menorrhagia (0.42 ± 0.04 g/L), 2 abortions (0.44 ± 0.04 g/L), and postpartum hemorrhage (0.33 ± 0.14 g/L) than in those without menorrhagia (0.46 ± 0.21 g/L, P = .60),abortion (0.47 ± 0.24 g/L, P = .85),and postpartum hemorrhage (0.50 ± 0.24 g/L, P = .16), respectively, but no significant differences were found.There was no significant difference in the Fg:C level between patients with (0.55 ± 0.03 g/L) and without postpartum thrombosis (0.47 ± 0.20 g/L, P = .42).In this family, the probands, his uncle, and his grandfather presented with bleeding from minor wounds (for more than 10 minutes) in their daily life.His mother suffered postpartum hemorrhage after cesarean delivery for her second child.The results were shown in Table 2.

| Clinical characteristics of patients with 3 types of CFDs
According to the levels of Fg:C and Fg:Ag and the fibrinogen gene analysis, a total of 3 patients were diagnosed with congenital hypofibrinogenemia (family 17; Supplementary Table S1 ), whereas the overall ISTH-BAT bleeding scores of bleeders in Hypodys-group (median, 2; range, 0-7) were similar to those in Dys-group (median, 2; range, 0-7; P > .05).No significant differences were observed for other variables between the 2 groups.
The results were shown in Table 3.  4).Three (3.2%) subjects with bleeding after tooth extraction received packing and or suture treatment.One (1.1%) subject with bleeding after mammary fibroma surgery received 500 mL of fresh frozen plasma (FFP) transfusion.

| Clinical management
Additionally, for the patient with a history of 2 spontaneous abortions, from the beginning of third pregnancy until delivery, antenatal management included a combination of fibrinogen infusions (40 mg/kg) to maintain fibrinogen plasma concentrations (Clauss) above 1 g/L and anticoagulant therapy.She eventually had a full-term vaginal delivery.
For the prophylaxis of thrombotic and 5 (5.4%) cases of menorrhagia received intermittent iron therapy.
There was no significant difference in the incidence of postoperative hemorrhage between highly and nonhighly fibrinolytic activity sites for patients with prophylactic treatment (P = .12),whereas for patients without prophylactic treatment, the incidence of postoperative hemorrhage at highly fibrinolytic activity sites (80%) was higher than that at nonhighly fibrinolytic activity sites (12%, P < .05).Overall, Fg:C levels were lower in patients who needed treatment (0.33 ± 0.14 g/L) than in those who did not (0.48 ± 0.25 g/L, P < .01),with the optimal Fg:C cutoff level of 0.44 g/L.
Notably, 1 girl with congenital Dys-, which was caused by 1 heterozygous missense mutation (c.991A>G) at the FGG exon 9, received hemostatic drug (tranexamic acid tablets) and treatment with 25 fibrinogen infusions (1 infusion per month with a dose of 30 mg/kg).
The menstrual period became shorter, but she still presents menometrorrhagia.Finally, after multidisciplinary (pediatric gynecology, pediatric hematology department, and clinical laboratory department) consultation, the girl received intrauterine device therapy to control the bleeding.The clinical effect of all 93 patients was considered very good in all events.

| D I S C U S S I O N
We have reported a retrospective analysis of 93 subjects with CFDs identified from 36 unrelated families.Currently, the laboratory diagnosis and classification of CFDs rely on detection of fibrinogen activity and antigen and then genetic evaluation [12,16,18,19].The 93 subjects exhibited prolonged TT, significantly decreased Fg:C, and normal or decreased Fg:Ag, which aligns with the previously proposed laboratory diagnosis for CFDs [14,16].
In our study, the main clinical association of CFDs was bleeding, which was identified in 57% subjects.Nevertheless, we recorded bleeding symptoms if they fulfilled the criteria for a bleeding score of ≥1 in the consensus ISTH-BAT [8,9], thus capturing mild bleeding episodes.Most subjects had bleeding at a single anatomical site, and only 24.7% of subjects had a bleeding score of ≥3.These findings suggest that most subjects with CFDs experienced mild bleeding, which is consistent with previous studies [20][21][22].Meanwhile, we found that the severity of bleeding symptoms was negatively correlated with the level of Fg:C (P < .0001),which is consistent with previous reports [20,23].mutations, c.510T>A (p.Asn170Lys) in the exon 4 of FGB [25] gene and c.902G>A (Arg301His) in the exon 8 of FGG gene, were identified as pathogenic mutations [21].Interestingly, the mutation c.510T>A (p.Asn170Lys) in FGB exon 4 was first identified in Chinese patients with congenital hypodysfibrinogenemia, which has been shown to affect the quality and quantity of the fibrinogen [25].Notably, a novel splice mutation, c.180+1G>A, at the boundary of intron 2-exon 2 of FGA was first detected in 4 Hypodys-patients with mild-to-moderate bleeding from the same family.We found that the FGA gene expression in splice mutation group was significantly lower than that in wildtype group (P < .05).Even though the number of cases of CFDs is already quite substantial in the study, only a small number of families underwent fibrinogen gene analysis, and more mutations will be identified in the future.
Currently, there is no standard or consensus on the treatments of CFDs.Replacement products in many countries are used for the treatment or prevention of bleeding in CFDs, including FFP, cryoprecipitates, or human fibrinogen concentrate [14,26,27].In our study, patients who needed treatment have lower Fg:C levels (P < .01)compared with those who did not.We recommended that prophylactic fibrinogen infusion should be properly considered for patients with Fg:C ≤0.44 g/L and for patients undergoing surgery at highly fibrinolytic activity sites.Fg:C levels that reached 1.05 g/L after infusion were efficacious and safe for on-demand treatment of bleeding and surgical prophylaxis.Because a small number of patients received fibrinogen infusions in the study, further clinical studies are needed to optimize the treatment guidelines for patients with CFDs.

12. 5
μL, the forward and reverse primers of 1 μL, respectively, ddH 2 O of 8 μL, and the DNA template of 2.5 μL.The amplification of PCR products was conducted under the following conditions: 5 minutes at 95 • C, 35 cycles for 40 seconds at 95 • C, 50 seconds at 55 • C, 1 minute at 72 • C, and 10 minutes at 72 • C. The purified PCR products were sent directly to Kindstar Global for sequencing, and the results were analyzed by Chromas software and NCBI GenBank.
The 30 women had 43 pregnancies, including 30 full-term vaginal births, 11 cesarean deliveries, and 2 early spontaneous abortions.The complete pregnancy medical history of 17 women was obtained.Noticeably, 1 patient had 2 consecutive abortions in the 8th and 11th week of pregnancy (autoimmune deficiency-induced miscarriage was excluded).There were 5 cases of postpartum hemorrhage, including 2 cases of vaginal delivery and 3 cases of cesarean delivery.Furthermore, 3 women had suffered from postpartum deep venous thrombosis (DVT), including 2 sisters (aged 36 and 38 years, family 10; All 93 subjects exhibited normal or slightly prolonged PT (mean, 12.6 seconds; range, 10.4-16.0seconds), normal aPTT (mean, 28.4 seconds; F I G U R E 1 Clinical characteristics of the 93 study subjects with congenital fibrinogen disorders.(A) Number of cases of different clinical symptoms in patients with congenital fibrinogen disorders.The number at the top of the bar chart refers to the number of cases of different bleeding episodes.(B) Ratios of different bleeding episodes in the total of 62 bleeding episodes of 53 patients.F I G U R E 2 Distribution of bleeding scores of 93 patients with congenital fibrinogen disorders by age.Bleeding scores (median, IQR) in subjects were determined using the ISTH bleeding assessment tool and were compared between the child and adult groups.ISTH-BAT, International Society on Thrombosis and Haemostasis bleeding assessment tool.range, 21.0-36.2seconds), significantly prolonged TT (mean, 29.2 seconds; range, 23.6-46.4seconds), significantly decreased Fg:C (mean, 0.45 g/L; range, 0.11-1.23 g/L), and normal or decreased Fg:Ag

A
total of 10 gene mutations were detected in 22 subjects from 8 unrelated pedigrees, including 1 homozygous missense mutation, 8 heterozygous missense mutations, and 1 splice mutation.Three compound missense mutations in the FGA, FGB, and FGG genes of 3 patients with congenital Hypodys-from the same family were identified.In this family, the sibling probands presented with moderate epistaxis, and their mother suffered postpartum hemorrhage after vaginal delivery for the first child.Furthermore, 1 girl with congenital Dys-, which was caused by 1 heterozygous missense mutation (c.991A>G) at the FGG exon 9, experienced menometrorrhagia (present since menarche and for more than 28 months) and menostaxis (prolonged lasting from 10 days to 1 month), whereas her mother was asymptomatic.Another heterozygous missense mutation (c.425T>G) at the FGB exon 3 was identified in 3 patients with congenital Hypo-from 1 family, including the probands with epistaxis, his asymptomatic elder brother, and his mother with bruises.Notably, the splice mutation c.180+1G>A at the junction of exon 2-intron 2 was first identified as a novel mutation in 4 patients with congenital Hypodys-from 1 family (HGMD, Clinvar database; https://site.geht.org/base-de-donnees-fibrinogene/).

F I G U R E 3
Distribution of bleeding episodes of 93 patients with congenital fibrinogen disorders by sex.(A) Number of cases of bleeding episodes at different anatomical sites between female and male groups.The number at the top of the bar chart refers to the total scores for different bleeding episodes.(B) Bleeding scores (median, IQR) were compared between the male and female groups.F I G U R E 4 Correlation analysis of fibrinogen activity (Fg:C) with the ISTH-BAT bleeding score.ISTH-BAT, International Society on Thrombosis and Haemostasis bleeding assessment tool.

5 |
C O N C L U S I O N S Most patients with CFDs have mild or no bleeding symptoms.Females with CFDs may face gynecologic and pregnancy complications.Fibrinogen activity combined with fibrinogen antigen and pedigree investigation can improve the feasibility and accuracy of diagnosis of CFDs.The severity of bleeding symptoms was negatively correlated with the levels of Fg:C.
Genotype and clinical phenotype in patients with congenital fibrinogen disorders (N = 22).
episodes, low-molecular-weight heparin at a dosage of 40 to 60 IE/kg was administered postpartum for 2 weeks.Five (5.4%) subjects with postpartum hemorrhage received 500-to 800-mL FFP transfusion (2 vaginal births and 3 cesarean deliveries).Two (2.2%) children with epistaxis received 1 to 2 fibrinogen infusions, with a daily dosage of 30 to 40 mg/kg.Thirty (3.2%) cases of menorrhagia received intermittent hormone therapy, T A B L E 2 Demographic and clinical characteristics of patients with 3 types of congenital fibrinogen disorders..0001(hypodysfibrinogenemia vs dysfibrinogenemia groups, due to a small number of cases of patients with hypofibrinogenemia).
a P < .05. b P < Type of operation, postoperative bleeding, and treatment of 39 patients with congenital fibrinogen disorders.
Our study reported 3 patients with hypodysfibrinogenemia from the same family who had 3 compound missense mutations in the FGA, FGB, and FGG genes.One of the mutations, c.991A>G (p.Thr331Ala) of FGA gene, was a known single nucleotide polymorphism without pathogenicity (https://www.ncbi.nlm.nih.gov/snp/rs6050).The other 2 T A B L E 4 TIAN ET AL.